Elimination of spores of Bacillus anthracis and other disease-causing bacteria from contaminated sites or materials is challenging due to the extreme resistance of spores to conventional antimicrobial treatments. Activation of the B. anthracis spore cortex peptidoglycan lytic machinery results in rapid loss of spore dormancy and resistance properties. This machinery can thus be targeted for germination-activating and spore-killing treatments. The cortex lytic enzyme SleB is stored in an inactive and highly stable form in the dormant spore and becomes active early during the germination process. The YpeB protein is required for incorporation of SleB into the dormant spore, and during germination, proteolytic cleavage of YpeB releases SleB to depolymerize the cortex. Aim 1 of the proposed work includes biochemical, genetic, and structural biology methods to reveal protein-protein contacts formed by YpeB and SleB in the dormant spore. Protein complexes containing YpeB or SleB will be purified from spores and co-purified proteins will be identified. Altered forms of YpeB will be expressed in vivo to identify key regions of the protein involved in function and protein interaction. The YpeB structure will be determined using X-ray crystallography in order to understand the effects of mutations. This work will clarify the mechanism of SleB stabilization, which may be adaptable to the production of stable protein pharmaceuticals, vaccines, and other industrial products. Aim 2 is the identification of the protease that processes YpeB and thus activates SleB. Knowledge of this protease and its regulation will suggest methods for the activation of germination via YpeB and SleB. The proposed work will contribute to two major translational goals: Activation of spore germination in order to improve decontamination and understanding of a mechanism of extreme protein stabilization.